A convenient method for producing the bleomycin-induced mouse model of scleroderma by weekly injections using a methylcellulose gel

Abstract

Systemic sclerosis (SSc) is a connective tissue disease characterized by vasculopathy, excessive accumulation of extracellular matrix, and fibrosis of the skin and internal organs. An animal model of SSc, the bleomycin-induced mouse model, has been established and used extensively to investigate the pathogenesis of SSc and to seek novel therapeutic agents. We recently developed thermo-reversible combination gels that can be injected subcutaneously and are made in aqueous solution by forming a complex coacervate with the substance of interest and cationic macromolecules, followed by co-formulation with methylcellulose (MC) as a negative thermosensitive polysaccharide. The objective of this study was to demonstrate whether weekly injections of bleomycin using combination gels loaded with bleomycin can induce the skin fibrosis model of SSc in susceptible mouse strains. A low molecular weight MC (4%) gel with 4.5% ammonium sulfate was made in aqueous solution, and mixed with bleomycin. This was injected subcutaneously into female C3H/He mice at weekly intervals. Control mice were injected with the gel made with phosphate-buffered saline. After 4 weeks, histological examination and gene expression assays of cytokines were performed. Examination in vitro showed that more than 80% of the bleomycin was released from the gel by the 4th day. Histological examination showed that dermal thickness increased in the MC-bleomycin-injected group compared with the control, and semi-quantitative analysis indicated that the extent of inflammation did not differ between the groups. In the MC-bleomycin-injected group, dermal fibrosis assessed with the Masson-Trichrome stain and numbers of alpha-smooth muscle actin-positive fibroblastic cells also increased. The procedure for inducing scleroderma in which bleomycin is injected weekly as an easily-made gel system using methylcellulose, can induce dermal fibrosis in susceptible mice without causing inflammation. We believe this system represents a time- saving and convenient procedure that should facilitate research on SSc.